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Abstract: In 2014 and 2015, synthetic cannabinoid receptor agonists NNEI (N‐1‐naphthalenyl‐1‐pentyl‐1H‐indole‐3‐carboxamide) and MN‐18 (N‐1‐naphthalenyl‐1‐pentyl‐1H‐indazole‐3‐carboxamide) were detected in recreationally used and abused products in multiple countries, and were implicated in episodes of poisoning and toxicity. Despite this, the pharmacokinetic profiles of NNEI and MN‐18 have not been characterized. In the present study NNEI and MN‐18 were incubated in rat and human liver microsomes and hepatocytes, to estimate kinetic parameters and to identify potential metabolic pathways, respectively. These parameters and pathways were then examined in vivo, via analysis of blood and urine samples from catheterized male rats following intraperitoneal (3 mg/kg) administration of NNEI and MN‐18. Both NNEI and MN‐18 were rapidly cleared by rat and human liver microsomes, and underwent a range of oxidative transformations during incubation with rat and human hepatocytes. Several unique metabolites were identified for the forensic identification of NNEI and MN‐18 intake. Interestingly, NNEI underwent a greater number of biotransformations (20 NNEI metabolites versus 10 MN‐18 metabolites), yet parent MN‐18 was eliminated at a faster rate than NNEI in vivo. Additionally, in vivo elimination was more rapid than in vitro estimates. These data highlight that even closely related synthetic cannabinoids can possess markedly distinct pharmacokinetic profiles, which can vary substantially between in vitro and in vivo models. [ABSTRACT FROM AUTHOR]

While allosteric modulators of the cannabinoid type-1 receptor (CB 1 ) continue to be developed and characterized, the gap between the in vitro and in vivo data is widening, raising questions regarding translatability of their effects and biological relevance. Among the CB 1 allosteric modulators, PSNCBAM-1 has received little attention regarding its effects in vivo. Recently, pregnenolone was reported to act as an allosteric modulator of CB 1 , blocking THC's effects in vitro and in vivo, highlighting the potential of CB 1 allosteric modulators for treatment of cannabis intoxication. We investigated the pharmacological effects of PSNCBAM-1 and two structural analogs, RTICBM-15 and -28, as well as pregnenolone, in both signaling and behavioral assays including [ 35 S]GTPγS binding, the cannabinoid tetrad and drug discrimination. While the CB 1 allosteric modulator PSNCBAM-1 attenuated THC-induced anti-nociception and its structural analog RTICBM-28 reduced THC's potency in drug discrimination, most cannabinoid effects in mice were unaffected. In contrast to the mouse studies, PSNCBAM-1 and analogs insurmountably antagonized CP55,940- and THC-stimulated [ 35 S]GTPγS binding and exhibited negative binding cooperativity with [ 3 H]SR141716 with similar apparent affinities. Notably, RTICBM-28, which contains a cyano substitution at the 4-chlorophenyl position of PSNCBAM-1, exhibited enhanced binding cooperativity with CP55,940. In contrast to previous findings, pregnenolone did not block THC's effects in drug discrimination or [ 35 S]GTPγS. These data further highlight the difficulty in translating pharmacological effects of CB 1 allosteric modulators in vivo but confirm the established pharmacology of PSNCBAM-1 and analogs in molecular assays of CB 1 receptor function. [ABSTRACT FROM AUTHOR]

Abstract: The endogenous cannabinoid system has been noted for its therapeutic potential, as well as the psychoactivity of cannabinoids such as Δ9-tetrahydrocannabinol (THC). However, less is known about the psychoactivity of anandamide (AEA), an endocannabinoid ligand. Thus, the goals of this study were to establish AEA as a discriminative stimulus in transgenic mice lacking fatty acid amide hydrolase (i.e., FAAH −/− mice unable to rapidly metabolize AEA), evaluate whether THC or oleamide, a fatty acid amide, produced AEA-like responding, and assess for CB1 mediation of AEA''s discriminative stimulus. Mice readily discriminated between 6mg/kg AEA and vehicle in a two-lever drug discrimination task. AEA dose-dependently generalized to itself. THC elicited full AEA-like responding, whereas oleamide failed to substitute. The CB1 antagonist rimonabant attenuated AEA- and THC-induced AEA-appropriate responding, demonstrating CB1 mediation of AEA''s discriminative stimulus. These findings suggest that, in the absence of FAAH, AEA produces intoxication comparable to THC, and consequently to marijuana. [Copyright &y& Elsevier]

Abstract: Cannabis sativa (marijuana plant) contains myriad cannabinoid compounds; yet, investigative attention has focused almost exclusively on Δ9-tetrahydrocannabinol (THC), its primary psychoactive substituent. Interest in modulation of THC''s effects by these other cannabinoids (e.g., cannabidiol (CBD)) has been stimulated anew by recent approval by Canada of Sativex (a 1:1 dose ratio combination of CBD:THC) for the treatment of multiple sclerosis. The goal of this study was to determine the degree to which THC''s abuse-related effects were altered by co-administration of CBD. To this end, CBD and THC were assessed alone and in combination in a two-lever THC discrimination procedure in Long-Evans rats and in a conditioned place preference/aversion (CPP/A) model in ICR mice. CBD did not alter the discriminative stimulus effects of THC at any CBD:THC dose ratio tested. In contrast, CBD, at CBD:THC dose ratios of 1:1 and 1:10, reversed CPA produced by acute injection with 10mg/kg THC. When administered alone, CBD did not produce effects in either procedure. These results suggest that CBD, when administered with THC at therapeutically relevant ratios, may ameliorate aversive effects (e.g., dysphoria) often associated with initial use of THC alone. While this effect may be beneficial for therapeutic usage of a CBD:THC combination medication, our discrimination results showing that CBD did not alter THC''s discriminative stimulus effects suggest that CBD:THC combination medications may also produce THC-like subjective effects at these dose ratios. [Copyright &y& Elsevier]

Background: The recent NIH mandate to consider sex as a biological variable in preclinical research has focused attention on delineation of sex differences in behavior. To investigate mechanisms underlying sex differences in Δ9-tetrahydrocannabinol (THC) effects, we examined the effects of sex and gonadal hormones on CB1 receptors in cerebellum, hippocampus, prefrontal cortex, and striatum.Methods: Adult Sprague-Dawley rats underwent gonadectomy (GDX) or sham-GDX. Half of the GDX females and males received estradiol or testosterone replacement (GDX+H), respectively. All rats were injected with vehicle or 30 mg/kg THC twice daily for 1 week before brain collection. CP55,940-stimulated [35S]GTPγS and [3H]SR141716A saturation binding assays were performed.Results: With exception of enhanced receptor activation in the hippocampi of female rats compared to males, vehicle-treated rats exhibited minimal sex differences in CB1 receptor densities or G-protein coupling. Repeated treatment with THC resulted in pronounced CB1 receptor desensitization and downregulation in both sexes in all brain regions with a greater magnitude of change in females.Conclusions: These results suggest that sex differences in the density and G-protein coupling of brain CB1 receptors may play a limited role in sex differences in acute THC effects not mediated by the hippocampus. In contrast, sex differences after repeated THC were common, with females (intact, GDX, and GDX+H) showing greater downregulation or desensitization in all four brain regions compared to the respective male groups. This result is consistent with a finding that women tend to progress to tolerance and dependence quicker than men after initiation of cannabis use. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoids (SCs) are novel psychoactive substances that are easily acquired, widely abused as a substitute for cannabis, and associated with cardiotoxicity and seizures. Although the structural bases of these compounds are scaffolds with known affinity and efficacy at the human cannabinoid type-1 receptor (hCB 1 ), upon ingestion or inhalation they can be metabolized to multiple chemical entities of unknown pharmacological activity. A large proportion of these metabolites are hydroxylated on the pentyl chain, a key substituent that determines receptor affinity and selectivity. Thus, the pharmacology of SC metabolites may be an important component in understanding the in vivo effects of SCs. We examined nine SCs (AB-PINACA, 5F-AB-PINACA, ADB/MDMB-PINACA, 5F-ADB, 5F-CUMYL-PINACA, AMB-PINACA, 5F-AMB, APINACA, and 5F-APINACA) and their hydroxypentyl (either 4-OH or 5-OH) metabolites in [ 3 H]CP55,940 receptor binding and the [ 35 S]GTP γ S functional assay to determine the extent to which these metabolites retain activity at cannabinoid receptors. All of the SCs tested exhibited high affinity (<10 nM) and efficacy for hCB 1 and hCB 2 The majority of the hydroxypentyl metabolites retained full efficacy at hCB 1 and hCB 2 , albeit with reduced affinity and potency, and exhibited greater binding selectivity for hCB 2 These data suggest that phase I metabolites may be contributing to the in vivo pharmacology and toxicology of abused SCs. Considering this and previous reports demonstrating that metabolites retain efficacy at the hCB 1 receptor, the full pharmacokinetic profiles of the parent compounds and their metabolites need to be considered in terms of the pharmacological effects and time course associated with these drugs., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB 1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB 1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB 1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB 1 agonist CP55,940 stimulated calcium mobilization and [ 35 S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [ 3 H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [ 3 H]CP55,940 and decrease binding of the antagonist [ 3 H]SR141716. In saturation binding studies, only increases in [ 3 H]CP55,940 B max , but not K d , were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [ 35 S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [ 3 H]CP5,5940 binding assay, indicating greater CB 1 receptor affinity and/or cooperativity.

Allosteric modulation of cannabinoid type-1 receptors (CB 1 ) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB 1 allosteric modulator yet there are conflicting reports regarding its effects on extracellular signal-regulated kinase (ERK) signaling. We conducted a systematic evaluation of Org27569 signaling through ERK. We have found that Org27569 is an antagonist of human CB 1 (hCB 1 ) mediated ERK signaling in HEK293 cells where it fully blocks CP55,940- but does not completely inhibit THC- and 2-AG-stimulated ERK1/2 activation. In hCB 1 HEK293 cells, CP55,940 (1 μM) treatment produced a significant increase in puncta at 20, 40, 60, and 120 min, consistent with receptor internalization. Org27569 (10 μM) co-treatment prevented internalization at each time point and alone had no effect. These data demonstrate that Org27569 can block the CP55,940- induced internalization of CB 1 receptors. Org27569 reduced basal ERK phosphorylation in hCB 1 HEK293 cells but not in untransfected cells, demonstrating that Org27569 acts via the CB 1 receptor to produce this effect. Furthermore, inverse agonism was through inhibition of G i/o as overnight treatment with pertussis toxin abated this response. Finally, to delineate Org27569's effects on ERK1/2 in subcellular compartments, subcellular fractionation was performed; Org27569 produced a significant decrease in ERK phosphorylation in the nuclear-enriched and cytosolic fractions. Altogether, these data are consistent with previous studies demonstrating that CB 1 -mediated ERK1/2 activation is G i/o -dependent and that Org27569 is an inverse agonist of CB 1 receptors. To our knowledge this is the first reported demonstration of inverse agonism of ERK signaling by Org27569., Competing Interests: Conflict of Interest: Authors have no conflict of interest concerning this study and have nothing to declare.